Panfungal PCR assays using fresh frozen paraffin embedded tissue specimens for fungal species identification and the detection of azole-resistance mutations in the A. fumigatus cyp51A gene at a South Korean hospital

Background: With rising concerns about changing fungal and azole resistance in identifying fungal species and patterns aspergillosis are crucial in the management of these diseases. The objectives of this study were to evaluate performance of panfungal PCR assays on formalin-fixed paraffin embedded (FFPE) samples for fungal species identification, and the detection of azole-resistance mutations in the Aspergillus fumigatus ( A.fumigatus ) cyp51A gene at a South Korean hospital. Methods: A total of 75 FFPE specimens with a histopathological diagnosis of aspergillosis or mucormycosis were identified during the 10-year study period (2006-2015). After deparaffinization and DNA extraction, panfungal PCR assays were conducted on FFPE samples for fungal species identification. The identified fungal species were compared with histopathological diagnosis. On samples identified as A.fumigatus , sequencings to identify frequent mutations in the cyp51A gene (tandem repeat 46 [TR46], L98H, and M220 alterations) that confer azole resistance were performed. Results: Specific fungal DNA was identified in 31 (41.3%) FFPE samples, and of these, 16 samples of specific fungal DNA were in accord with histopathological diagnosis of aspergillosis or mucormycosis. 15 samples had discordant histopathology and PCR results. No azole-mediating cyp51A gene mutation was revealed among nine cases of A. fumigatus . Moreover, no cyp51A mutations were identified among three cases with history of prior azole use. Conclusion: The pan-fungal PCR assay with FFPE sample may provide additional information on fungal species identification. No azole-resistance mediating mutations in the A. fumigatus cyp51A gene were identified among FFPE samples during study period.


Background
Mucormycosis (formerly known as zygomycosis) and aspergillosis are invasive fungal diseases that usually present as rhino-orbital-cerebral or pulmonary infections. [1,2] Aspergillus species are usually susceptible to voriconazole, and isavuconzole has also become a first-line targeted therapy [3], whereas voriconazole has no activity agents mucorales. [3] Moreover, concerns about changing epidemiology and azole resistance are rising.
Higher rates of mortality have been demonstrated for patients treated with voriconazole in voriconazole-resistant invasive aspergillosis (IA) than for voriconazole-susceptible IA [4,5]. Rapid detection of fungal species and of azole-resistance in Aspergillosis fumigatus (A. fumigatus) may benefit outcomes by guiding appropriate antifungal therapy. [4] Azoles are inhibitors of 14α-sterol demethylases, which are responsible for catalyzing a critical step in the biosynthesis of ergosterol a component of fungal membrane.
[5] Mutation in the cyp51A gene, which is responsible for encoding 14α-sterol demethylase enzymes, is the most common azoleresistance mechanism in Aspergillus species.
[6] Moreover, isolates harboring tandem repeats (TRs) in the promoter region of the cyp51A gene and point mutations leading to amino acid changes are also known to cause azole-resistance.
[6] Furthermore, the incidence of azole-resistant Aspergillus species has increased over recent years due to previous exposure and environment-associated resistance. [5,7] Molecular methods can now be used to rapidly identify fungal species in Formalin-Fixed and Para Mucormycosis (formerly known as zygomycosis) and aspergillosis are invasive fungal diseases that usually present as rhino-orbital-cerebral or pulmonary infections. [1,2] Aspergillus species are usually susceptible to voriconazole, and isavuconzole has also become a first-line targeted therapy [

Results
During the 10-year study period, 75 patients received a histopathological diagnosis of mucormycosis or aspergillosis, and PCR amplification and identification was positive for 31 (41.3%) of the 75 FFPE samples.
Sixteen FFPE samples had corresponding histopathology and PCR sequencing results. Fourteen cases of Aspergillus species were identified; A. fumigatus (n = 9), A. flavus (n = 2), A. oryzae (n = 2), and A. tamarii (n = 1). Two cases with a histopathological diagnosis of mucormycosis were identified as Rhizopus oryzae by sequence analysis. The nine cases identified as A. fumigatus species were further analyzed for azole-resistance mutations in the A. fumigatus cyp51A gene.
The demographic and clinical data of the 16 patients identified as aspergillosis or mucormycosis by panfungal PCR are presented in Table 1. Patient 1, a 77-year-old male, had a history of chronic obstructive pulmonary disease (COPD) and was receiving steroids when he developed a brain abscess. Empirical antibacterial agent, but no antifungal agent, was administered. Aspergillosis was confirmed after death by pathologic diagnosis, and PCR sequencing confirmed A. fumigatus. Patient 2 was an 81-year-old male patient who developed fungal pneumonia after surgery for renal cell carcinoma. Pathologic diagnosis conducted on transbronchial lung biopsy tissue revealed aspergillosis. The patient was treated with voriconazole but succumbed despite appropriate treatment. PCR sequencing identified A. fumigatus. Patient 4 was a 75-year-old male patient who had undergone liver transplantation due to hepatocellular carcinoma and was receiving immunosuppressive therapy and oral itraconazole for fungal prophylaxis prior to developing acute maxillary sinusitis. Patient 5 was a 62-year-old female and had undergone liver transplantation due to The results of the cyp51A alterations of the nine samples confirmed as A. fumigatus are summarized in table 1. Seven samples were positive by the L98H PCR assay alone, but no mutations were detected by sequence analysis. Seven samples were positive by the M220 PCR assay alone, but also revealed no mutations by sequence analysis. Six samples were positive by the TR 46 PCR assay alone, but similarly, no mutations were revealed by sequence analysis.
Discordant PCR and histopathology results were obtained for 15 samples. Two specimens histopathologically diagnosed as Aspergillosis were identified as mucorales; Lichtheimia ramose and Rhizopus oryzae. Five specimens with a histopathological diagnosis of aspergillosis were identified by PCR as Epicoccum nigrum, Bipolaris zeicola, Fusarium solani, Nakataea oryzae and Cladosporium cladosporioides. Eight samples were identified by PCR sequencing as uncultured fungus clones. One brain sample diagnosed as mucormycosis by histopathology was identified by PCR as an uncultured fungus clone. Lichtheimia ramose was identified by PCR in buttock tissue. Rhizopus oryzae, Bipolaris zeicola, Fusarium solani, Nakataea oryzae were all identified in sinus samples with uncultured fungus clones, and Epicoccum nigrum and Cladosporium cladosporioides were identified in lung samples.

Discusison
The identification of fungal DNA in tissue samples by PCR improves diagnostic accuracies for fungal infections [3], and pan-fungal PCR conducted on FFPE tissues provides an alternative to culture dependent identification methods.
[8] Mucorales has been identified by PCR in paraffin-embedded tissue samples of patients with a fungal infection [11,12], and it has been shown fungal organisms can be identified by amplifying fungal ITS 1 and 2 using pan-fungal primers. [12] The results of the present study concur that PCR amplification of the ITS 1 and 2 regions accurately diagnoses fungal species in FFPE specimens.
In all 31 FFPE samples that produced amplifiable DNA results, fungi were identified to the genus or species level. In 2 cases with a histopathologic diagnosis of aspergillosis, mucorale specific DNA was identified by sequencing PCR products. Although this may have been due to tissue specimen contamination, the risk of misdiagnosis by histopathology cannot be excluded. Similar cases have been described in cases confirmed by culture. [13] Two samples with a histopathologic diagnosis of aspergillosis were identified as mucorales; Lichtheimia ramose and Rhizopus oryzae. The Lichtheimia species (formerly known as Absidia) are currently regarded as emerging pathogens among Mucoralean fungi. [14] In the present study, the male patient identified with Lichtheimia ramose infection had a history of hepatocellular carcinoma and had undergone liver transplantation prior to infection. In addition, he was under immunosuppressive medication. Biopsy from a buttock revealed mucormycosis by PCR product sequencing. Although it is generally known to have low virulence, cases of mucormycosis due to Lichtheimia ramose in immunocompromised hosts have been reported. [14,15] Chaumont et al.
reported a case of cutaneous mucormycosis requiring aggressive surgical debridement. [16] The second case, initially diagnosed by histopathology as aspergillosis, was found to be due to Rhizopus oryzae by PCR. This patient had a history of aplastic anemia before fungal infection and displayed rapid clinical deterioration resulting in death. Rhizopus oryzae is the most common cause of zygomycosis, and is a life-threatening infection that usually occurs in patients with diabetic ketoacidosis. [17] Four samples histopathologically diagnosed as Aspergillosis produced ambiguous results.  and TR46/Y121F/T289A are commonly associated with azole-resistance linked to environmental use of azoles in agriculture, and often found in azole-naïve patients. [26,27] In the present study, azole resistance was not detected in three cases (patients 4, 5, and 9) with a history of prior azole use.
Thus, because sample numbers were small, we suggest larger scale studies be performed to investigate azole resistance in patients with a history of azole exposure.
Several limitations of the present study warrant mention. First, the amount of fungal DNA available is crucial when investigating clinical samples, and DNA degradation and the effects of formaldehyde may have reduced DNA amounts in samples. Second, culture results or azole susceptibility profiles were not considered. Third, as the TR46 and M220 mutations have never been reported in South Korea, a positive control for isolates harboring these mutations could not be acquired. And finally, there are many mutations within cyp51A that can confer elevated MICs/resistance to the triazoles, not just the ones stated in this paper, as well there areas unknown methods of elevated MICs to the triazoles not linked to cyp51A mutations that would not be detected by this assay.

Conclusion
The pan-fungal PCR assay with FFPE sample may provide additional information on fungal species identification. No azole-resistance mediating mutations in the A. fumigatus cyp51A gene were identified among FFPE samples during study period.

PCR assays and controls
To amplify ITS regions, PCR was performed in total volumes of 50 μl, consisting of 1X reaction buffer, 0.1 μM dNTPmix, 1.25 U of Taq DNA Polymerase (RBC Bioscience, Xindian City, Taiwan), 20 pmol of each primer, and 200 ng of DNA (1 μL) per sample. PCR was performed using the following protocol; 95°C for 3 minutes, 35 amplification cycles of 94°C for 30 seconds, 50°C for 1 minute, and 72°C for 1 minute, and a final extension at 72°C for 7 minutes.
To detect L98H and M220 alterations, PCR was conducted in a total volume of 50 μl containing 2 μl template DNA (100 ng human DNA + unknown amount of A. fumigatus DNA), 1X reaction buffer, 0.1 μM dNTPmix, 1.25 U of Taq DNA Polymerase (RBC Bioscience, Xindian City, Taiwan), and 20 pmol of each primer. The PCR amplification protocol was as follows; 5 min of initial denaturation at 94°C, 39 amplification cycles of 94°C for 45 s, 52°C for 1 min, and 72°C for 1 min, and final extension at 72°C for 10 min.
To detect TR46 alterations, PCR was conducted in a total volume of 50 µl as described for L98H and M220 above. The PCR amplification protocol was as follows; 5 min denaturation at 94°C, 22 amplification cycles of 94°C for 45 s, 52°C for 1 min, and 72°C for 1 min, and final extension at 72°C for 5 min. For the second PCR step, we used a total volume of 50 μl and 3 μl of the first-step PCR mixture as template. Other components were as described for L98H and M220. The second step PCR amplification protocol was as follows; 5 min initial denaturation at 94°C, 34 amplification cycles of 94°C for 45s, 56°C for 1 min, and 72°C for 1 min, and final extension at 72°C for 5 min.

Sequence analysis
To identify Aspergillus species and mucorales, PCR products were purified using the MiniElute PCR purification kit (Qiagen, Hilden, Germany). A minimum of 50 ng DNA was sequenced using the BigDye Terminator version 3.1 Cycle Sequencing kit (Applied Biosystems, Foster City, CA) and an Applied Biosystems 3730XL DNA Analyzer (Applied Biosystems, Foster City, CA). Sequences were edited and aligned using Sequence Scanner Software 2 ver. 2.0 (Applied Biosystems, Foster City, CA), and product sequences were compared with reference sequences using the NCBI alignment service AlignSequenNucleotideBlast (http://www.ncbi.nlm.nih.gov/). The GenBank accession number for the A.
fumigatus sequences determined in this study is CM000169.1.
To detect potential mutations in the PCR products subjected to DNA sequence analysis, sequences were compared with the A. fumigatus cyp51A wild-type sequence using the NCBI alignment service AlignSequenceNucleotideBlast (http://www.ncbi.nlm.nih.gov/). burden in brain measured by quantitative PCR, and improves survival at a low but not a high dose during murine disseminated zygomycosis.